Effects of N-acylethanolamines on the respiratory chain and production of reactive oxygen species in heart mitochondria

Long-chain N-acylethanolamines (NAEs) have been found to uncouple oxidative phosphorylation and to inhibit uncoupled respiration of rat heart mitochondria [Wasilewski, M., Wieckowski, M.R., Dymkowska, D. and Wojtczak, L. (2004) Biochim. Biophys. Acta 1657, 151-163]. The aim of the present work was to investigate in more detail the mechanism of the inhibitory effects of NAEs on the respiratory chain. In connection with this, we also investigated a possible action of NAEs on the generation of reactive oxygen species (ROS) by respiring rat heart mitochondria. It was found that unsaturated NAEs, N-oleoylethanolamine (N-Ole) and, to a greater extent, N-arachidonoylethanolamine (N-Ara), inhibited predominantly complex I of the respiratory chain, with a much weaker effect on complexes II and III, and no effect on complex IV. Saturated N-palmitoylethanolamine had a much smaller effect compared to unsaturated NAEs. N-Ara and N-Ole were found to decrease ROS formation, apparently due to their uncoupling action. However, under specific conditions, N-Ara slightly but significantly stimulated ROS generation in uncoupled conditions, probably due to its inhibitory effect on complex I. These results may contribute to our better understanding of physiological roles of NAEs in protection against ischemia and in induction of programmed cell death.     

Spatio-temporal relief from hypoxia and production of reactive oxygen species during bud burst in grapevine (Vitis vinifera)

Background and Aims Plants regulate cellular oxygen partial pressures (pO₂), together with reduction/oxidation (redox) state in order to manage rapid developmental transitions such as bud burst after a period of quiescence. However, our understanding of pO₂ regulation in complex meristematic organs such as buds is incomplete and, in particular, lacks spatial resolution.Methods The gradients in pO₂ from the outer scales to the primary meristem complex were measured in grapevine (Vitis vinifera) buds, together with respiratory CO₂ production rates and the accumulation of superoxide and hydrogen peroxide, from ecodormancy through the first 72 h preceding bud burst, triggered by the transition from low to ambient temperatures.Key Results Steep internal pO₂ gradients were measured in dormant buds with values as low as 2·5 kPa found in the core of the bud prior to bud burst. Respiratory CO₂ production rates increased soon after the transition from low to ambient temperatures and the bud tissues gradually became oxygenated in a patterned process. Within 3 h of the transition to ambient temperatures, superoxide accumulation was observed in the cambial meristem, co-localizing with lignified cellulose associated with pro-vascular tissues. Thereafter, superoxide accumulated in other areas subtending the apical meristem complex, in the absence of significant hydrogen peroxide accumulation, except in the cambial meristem. By 72 h, the internal pO₂ gradient showed a biphasic profile, where the minimum pO₂ was external to the core of the bud complex.Conclusions Spatial and temporal control of the tissue oxygen environment occurs within quiescent buds, and the transition from quiescence to bud burst is accompanied by a regulated relaxation of the hypoxic state and accumulation of reactive oxygen species within the developing cambium and vascular tissues of the heterotrophic grapevine buds.     

AIF,reactive oxygen species,and neurodegeneration: A “complex” problem

Apoptosis-inducing factor (AIF) is a flavin-binding mitochondrial intermembrane space protein that is implicated in diverse but intertwined processes that include maintenance of electron transport chain function, reactive oxygen species regulation, cell death, and neurodegeneration. In acute brain injury, AIF acquires a pro-death role upon translocation from the mitochondria to the nucleus, where it initiates chromatin condensation and large-scale DNA fragmentation. Although harlequin mice exhibiting an 80–90% global reduction in AIF protein are resistant to numerous forms of acute brain injury, they paradoxically undergo slow, progressive neurodegeneration beginning at three months of age. Brain deterioration, accompanied by markers of oxidative stress, is most pronounced in the cerebellum and retina, although it also occurs in the cortex, striatum, and thalamus. Loss of an AIF pro-survival function linked to assembly or stabilization of electron transport chain complex I underlies chronic neurodegeneration. To date, most studies of neurodegeneration have failed to adequately separate the relative importance of the mitochondrial and nuclear functions of AIF in determining the extent of injury, or whether oxidative stress plays a causative role. This review explores the complicated relationship among AIF, complex I, and the regulation of mitochondrial reactive oxygen species levels. It also discusses the controversial role of complex I deficiency in Parkinson’s disease, and what can be learned from the AIF- and complex I-depleted harlequin mouse.     

Zinc induces distinct changes in the metabolism of reactive oxygen and nitrogen species (ROS and RNS) in the roots of two Brassica species with different sensitivity to zinc stress

Background and Aims Zinc (Zn) is an essential micronutrient naturally present in soils, but anthropogenic activities can lead to accumulation in the environment and resulting damage to plants. Heavy metals such as Zn can induce oxidative stress and the generation of reactive oxygen and nitrogen species (ROS and RNS), which can reduce growth and yield in crop plants. This study assesses the interplay of these two families of molecules in order to evaluate the responses in roots of two Brassica species under high concentrations of Zn.Methods Nine-day-old hydroponically grown Brassica juncea (Indian mustard) and B. napus (oilseed rape) seedlings were treated with ZnSO₄ (0, 50, 150 and 300 µm) for 7 d. Stress intensity was assessed through analyses of cell wall damage and cell viability. Biochemical and cellular techniques were used to measure key components of the metabolism of ROS and RNS including lipid peroxidation, enzymatic antioxidants, protein nitration and content of superoxide radical (O2·−), nitric oxide (NO) and peroxynitrite (ONOO⁻).Key Results Analysis of morphological root damage and alterations of microelement homeostasis indicate that B. juncea is more tolerant to Zn stress than B. napus. ROS and RNS parameters suggest that the oxidative components are predominant compared with the nitrosative components in the root system of both species.Conclusions The results indicate a clear relationship between ROS and RNS metabolism as a mechanism of response against stress caused by an excess of Zn. The oxidative stress components seem to be more dominant than the elements of the nitrosative stress in the root system of these two Brassica species.     

Spatial and temporal regulation of the metabolism of reactive oxygen and nitrogen species during the early development of pepper (Capsicum annuum) seedlings

Background and Aims The development of seedlings involves many morphological, physiological and biochemical processes, which are controlled by many factors. Some reactive oxygen and nitrogen species (ROS and RNS, respectively) are implicated as signal molecules in physiological and phytopathological processes. Pepper (Capsicum annuum) is a very important crop and the goal of this work was to provide a framework of the behaviour of the key elements in the metabolism of ROS and RNS in the main organs of pepper during its development.Methods The main seedling organs (roots, hypocotyls and green cotyledons) of pepper seedlings were analysed 7, 10 and 14 d after germination. Activity and gene expression of the main enzymatic antioxidants (catalase, ascorbate–glutathione cycle enzymes), NADP-generating dehydrogenases and S-nitrosoglutathione reductase were determined. Cellular distribution of nitric oxide (^·NO), superoxide radical (O₂^·–) and peroxynitrite (ONOO^–) was investigated using confocal laser scanning microscopy.Key Results The metabolism of ROS and RNS during pepper seedling development was highly regulated and showed significant plasticity, which was co-ordinated among the main seedling organs, resulting in correct development. Catalase showed higher activity in the aerial parts of the seedling (hypocotyls and green cotyledons) whereas roots of 7-d-old seedlings contained higher activity of the enzymatic components of the ascorbate glutathione cycle, NADP-isocitrate dehydrogenase and NADP-malic enzyme.Conclusions There is differential regulation of the metabolism of ROS, nitric oxide and NADP dehydrogenases in the different plant organs during seedling development in pepper in the absence of stress. The metabolism of ROS and RNS seems to contribute significantly to plant development since their components are involved directly or indirectly in many metabolic pathways. Thus, specific molecules such as H₂O₂ and NO have implications for signalling, and their temporal and spatial regulation contributes to the success of seedling establishment.     

黄皮种子线粒体呼吸速率和活性氧清除酶活性对脱水的响应及其生态学意义

顽拗性种子脱落时具有较高的含水量和代谢活性, 对脱水高度敏感; 但顽拗性种子脱水敏感性的机理至今仍然不清楚。该文以顽拗性黄皮(Clausena lansium)种子为材料, 研究了种子和胚轴对水分丧失的响应, 在脱水过程中胚轴和子叶的呼吸速率, 胚轴和子叶线粒体的细胞色素c氧化酶(CCO)活性、外膜完整性、CCO和交替氧化酶(AOX)途径以及线粒体活性氧清除酶活性的变化。结果表明, 随着水分的丧失, 种子和胚轴的存活率逐渐下降, 种子的脱水敏感性大于胚轴; 胚轴和子叶的呼吸速率以及线粒体外膜的完整性降低。胚轴和子叶线粒体的CCO途径以及胚轴AOX途径的呼吸速率在脱水初期增加, 随着继续脱水下降, 胚轴线粒体AOX途径的呼吸速率则随着脱水显著下降。胚轴线粒体的超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)和谷胱甘肽还原酶(GR)活性和子叶线粒体的APX活性随着脱水迅速下降; 胚轴线粒体的脱氢抗坏血酸还原酶(DHAR)活性和子叶线粒体的SOD、DHAR和GR活性在脱水初期增加, 然后下降。这些数据表明黄皮种子的脱水敏感性与线粒体的呼吸速率和活性氧清除酶的活性降低密切相关, 也与长期适应热带/亚热带的生境有关。     

The effect of electromagnetic field on reactive oxygen species production in human neutrophils in vitro

The present study was undertaken in order to determine the effect of low frequency electromagnetic field (EMF) on reactive oxygen species (ROS) production in human neutrophils in peripheral blood in vitro. We investigated how differently generated EMF and several levels of magnetic induction affect ROS production. To evaluate the level of ROS production, two fluorescent dyes were used: 2′7′-dichlorofluorscein-diacetate and dihydrorhodamine. Phorbol 12-myristate 13-acetate (PMA), known as strong stimulator of the respiratory burst, was also used. Alternating magnetic field was generated by means of Viofor JPS apparatus. Three different levels of magnetic induction have been analyzed (10, 40 and 60 μT). Fluorescence of dichlorofluorescein and 123 rhodamine was measured by flow cytometry. The experiments demonstrated that only EMF tuned to the calcium ion cyclotron resonance frequency was able to affect ROS production in neutrophils. Statistical analysis showed that this effect depended on magnetic induction value of applied EMF. Incubation in EMF inhibited cell activity slightly in unstimulated neutrophils, whereas the activity of PMA-stimulated neutrophils has increased after incubation in EMF.     

Cellular reactive oxygen species inhibitory constituents of Hypericum thasium Griseb

The phytochemical investigation of the ethyl acetate extract of Hypericum thasium has led to the characterization of four benzophenone derivatives 1-4, a known benzophenone 5 and four known flavonoids, quercetin (6), quercitrin (7), isoquercetin (8), and 3, 8′′-biapigenin (9). Lucigenin- and luminal-based chemiluminescence assays were employed to monitor the inhibitory activity of these compounds towards the production of reactive oxygen species (ROS) by human polymorphoneutrophils (PMNs). The assay results showed that benzophenones 1 and 3 are extracellular inhibitors of ROS production, while flavonoids 6, 8, and 9 can modulate intracellular ROS production.     

Auxin-induced reactive oxygen species production requires the activation of phosphatidylinositol 3-kinase

We recently reported that production of reactive oxygen species (ROS) is essential for auxin-induced gravitropic signaling. Here, we investigated the role of phosphatidylinositol 3-kinase and its product, PtdIns(3)P, in auxin-mediated ROS production and the root gravitropic response. Pretreatment with LY294002, an inhibitor of PtdIns 3-kinase activity, blocked auxin-mediated ROS generation, and reduced the sensitivity of root tissue to gravistimulation. The amount of PtdIns(3)P increased in response to auxin, and this effect was abolished by pretreatment with LY294002. In addition, sequestration of PtdIns(3)P by transient expression of the endosome binding domain in protoplasts abrogated IAA-induced ROS accumulation. These results indicate that activation of PtdIns 3-kinase and its product PtdIns(3)P are required for auxin-induced production of ROS and root gravitropism.     

Schistosoma mansoni: ferredoxin-NADP(H) oxidoreductase and the metabolism of reactive oxygen species

Mitochondrial-type ferredoxin-NADP(H) oxidoreductases (FNR) catalyze the electron transport between NADPH and substrates such as ferredoxins. Even though enzymes belonging to this family are present in several organisms, including prokaryotes, their biological function is not clearly understood. In a previous work, we reported the existence of a mitochondrial-type FNR in the trematode Schistosoma mansoni (SmFNR). This enzyme conferred tolerance to oxidative stress conditions when tested in an heterologous system. In this work, we demonstrate that the SmFNR can be imported to mitochondria in mammal cells and show that its expression is induced in parasite cultures by reactive oxygen species (ROS). The results reported herein give further support to the involvement of SmFNR in ROS metabolism.     

Effect of lanthanum ions (La3+) on the reactive oxygen species scavenging enzymes in wheat leaves

Physiological effects of lanthanum ions on the activities of the enzymes in the reactive oxygen species (ROS) scavenging system in leaves of wheat (Triticum aestivum L.) seedlings were studied. Wheat leaves treated in Hogland solution with 0.1 mM LaCl₃ for 48 h showed increased levels of superoxide dismutase (SOD), catalase (CAT), ascorbate-specific peroxidase (AsA-POD), and dehydroascorbate reductase (DHAR). However, a minor effect was observed on the levels of monodehydroascorbate reductase (MDAR) and glutathione reductase (GR), which regulate the release of energy required by the ROS scavenging system. The whole system was linked up by H⁺ transmission. Our results indicated that the activities of the enzymes that function directly to remove ROS were elevated by La³⁺ treatment, which is consistent with the observations that La³⁺-treated plants had increased tolerance to environmental stresses. The remaining levels of MDAR and GR suggested that these two enzymes might be regulated differently from that of the other four enzymes studied.     

The impact of reactive oxygen species and genetic mitochondrial mutations in Parkinson's disease

The exact pathogenesis of Parkinson's disease (PD) is still unknown and proper mechanisms that correspond to the disease remain unidentified. It is understood that PD is age-related; as age increases, the chance of onset responds accordingly. Although there are no current means of curing PD, the understanding of reactive oxygen species (ROS) provides significant insight to possible treatments. Complex I deficiencies of the respiratory chain account for the majority of unfavorable neural apoptosis generation in PD. Dopaminergic neurons are severely damaged as a result of the deficiency. Symptoms such as inhibited cognitive ability and loss of smooth motor function are the results of such impairment. The genetic mutations of Parkinson's related proteins such as PINK1 and LRRK2 contribute to mitochondrial dysfunction which precedes ROS formation. Various pathways are inhibited by these mutations, and inevitably causing neural cell damage. Antioxidants are known to negate the damaging effects of free radical overexpression. This paper expands on the specific impact of mitochondrial genetic change and production of free radicals as well as its correlation to the neurodegeneration in Parkinson's disease.     

Anionic phospholipid-induced regulation of reactive oxygen species production by human cytochrome P450 2E1

We suggest that the cytochrome P450 2E1 (CYP2E1)-induced formation of reactive oxygen species (ROS) can be regulated by anionic phospholipids and the presence of the N-terminal region of the enzyme. When the content of cardiolipin (CL) in membranes at the expense of phosphatidylcholine matrix was increased, the ROS produced by recombinant human CYP2E1 was decreased as a function of CL concentration. On the contrary, the N-terminally truncated CYP2E1 had a decreased effect on the lipid-induced reduction of ROS formation. These results suggest that specific phospholipids can regulate the function of CYP2E1 by interaction with the enzyme including the N-terminal region(s).     

An example of molecular co-evolution: reactive oxygen species (ROS) and ROS scavenger levels in Schistosoma mansoni/Biomphalaria glabrata interactions

The co-evolution between hosts and parasites involves huge reciprocal selective pressures on both protagonists. However, relatively few reports have evaluated the impact of these reciprocal pressures on the molecular determinants at the core of the relevant interaction, such as the factors influencing parasitic virulence and host resistance. Here, we address this question in a host-parasite model that allows co-evolution to be monitored in the field: the interaction between the mollusc, Biomphalaria glabrata, and its trematode parasite, Schistosoma mansoni. Reactive oxygen species (ROS) produced by the haemocytes of B. glabrata are known to play a crucial role in killing S. mansoni. Therefore, the parasite must defend itself against oxidative damage caused by ROS using ROS scavengers in order to survive. In this context, ROS and ROS scavengers are involved in a co-evolutionary arms race, and their respective production levels by sympatric host and parasite could be expected to be closely related. Here, we test this hypothesis by comparing host oxidant and parasite antioxidant capabilities between two S. mansoni/B. glabrata populations that have co-evolved independently. As expected, our findings show a clear link between the oxidant and antioxidant levels, presumably resulting from sympatric co-evolution. We believe this work provides the first supporting evidence of the Red Queen Hypothesis of reciprocal evolution for functional traits at the field-level in a model involving a host and a eukaryotic parasite.     

Increase of reactive oxygen species in different tissues during lipopolysaccharide-induced fever and antipyresis: an electron paramagnetic resonance study

Abstract

Fever is a regulated increase in body temperature and a component of the acute-phase response, triggered mainly after the invasion of pathogens in the body. Reactive oxygen species (ROS) are generated during the physiological and pathological processes, and can act as both signalling molecules as well as promoters of oxidative stress. Male Wistar rats, pretreated with oral doses of acetaminophen, celecoxib, dipyrone, or ibuprofen 30?min before an intravenous lipopolysaccharide (LPS) or sterile saline injection, showed a reduced febrile response in all animals tested. The formation of ROS in the fresh blood, liver, brown adipose tissue (BAT), and hypothalamus of febrile and antipyretic-treated animals was assessed by electron paramagnetic resonance using the spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH). While the CM^? concentrations remained unaltered in the blood samples examined 5?h after the induction of fever, we found increased CM^? levels in the liver (in µM, saline: 290?±?42; LPS: 512?±?34), BAT (in µM, saline: 509?±?79, LPS: 855?±?79), and hypothalamus (in µM, saline: 292?±?35; LPS: 467?±?8) at the same time point. Importantly, none of the antipyretics were seen to alter the CM^? accumulation profile. Data from this study suggest that there is an increased formation of ROS in the different tissues during fever, which may cause oxidative stress, and that the antipyretics tested do not interfere with ROS production.     

The role of reactive oxygen and nitrogen species in cellular iron metabolism

The catalytic role of iron in the Haber-Weiss chemistry, which results in propagation of damaging reactive oxygen species (ROS), is well established. In this review, we attempt to summarize the recent evidence showing the reverse: That reactive oxygen and nitrogen species can significantly affect iron metabolism. Their interaction with iron-regulatory proteins (IRPs) seems to be one of the essential mechanisms of influencing iron homeostasis. Iron depletion is known to provoke normal iron uptake via IRPs, superoxide and hydrogen peroxide are supposed to cause unnecessary iron uptake by similar mechanism. Furthermore, ROS are able to release iron from iron-containing molecules. On the contrary, nitric oxide (NO) appears to be involved in cellular defense against the iron-mediated ROS generation probably mainly by inducing iron removal from cells. In addition, NO may attenuate the effect of superoxide by mutual reaction, although the reaction product—peroxynitrite—is capable to produce highly reactive hydroxyl radicals.     

Effect of stationary magnetic field strengths of 150 and 200 mT on reactive oxygen species production in soybean

Our previous investigation reported the beneficial effect of pre-sowing magnetic treatment for improving germination parameters and biomass accumulation in soybean. In this study, soybean seeds treated with static magnetic fields of 150 and 200 mT for 1 h were evaluated for reactive oxygen species (ROS) and activity of antioxidant enzymes. Superoxide and hydroxyl radicals were measured in embryos and hypocotyls of germinating seeds by electron paramagnetic resonance spectroscopy and kinetics of superoxide production; hydrogen peroxide and antioxidant activities were estimated spectrophotometrically. Magnetic field treatment resulted in enhanced production of ROS mediated by cell wall peroxidase while ascorbic acid content, superoxide dismutase and ascorbate peroxidase activity decreased in the hypocotyl of germinating seeds. An increase in the cytosolic peroxidase activity indicated that this antioxidant enzyme had a vital role in scavenging the increased H(2)O(2) produced in seedlings from the magnetically treated seeds. Hence, these studies contribute to our first report on the biochemical basis of enhanced germination and seedling growth in magnetically treated seeds of soybean in relation to increased production of ROS.     

Bimodal actions of reactive oxygen species in the differentiation and bone-resorbing functions of osteoclasts

In order to demonstrate that cellular redox status undergoes decreased reduction during osteoclast differentiation and further decreased reduction during osteoclastic bone resorption, we analyzed gamma-glutamylcysteinyl synthetase activity, a glutathione synthesis rate-limiting enzyme, and total glutathione and thiol groups. Moderate and severe redox shifts towards a more oxidizing environment induced gradual increases and decreases in osteoclastogenesis. Moreover, while severe glutathione depletion inhibited bone resorption, moderate glutathione repletion enhanced bone resorption. In summary, our observations suggest that there is a threshold for redox status, representing biphasic patterns in osteoclast differentiation and function.     

The cell cycle related apoptotic susceptibility to arsenic trioxide is associated with the level of reactive oxygen species

Double staining flow cytometry was performed using 7-amino actinomycin D and 6-carboxy-2‘,7‘-dichlorodihydrofluorescein diacetate, to detect the level fluctuation of reactive oxygen species (ROS) during the cell cycle of normal NB4 cells. Our results showed that NB4 cells possessed higher level of ROS in G2/M phase than in G1 and S phases. Double staining flow cytometry, with TdT mediated dUTP nick end labeling (Tunel) and propidium iodide(PI), indicated that As2O3 (2μM) could induce apoptosis in NB4 cells prevailingly from G2/M phase, and this efficacy was enhanced upon co-administration of 2, 3-dimethoxy-1, 4-naphthoquinone (DMNQ) (2.5μM) which could produce the endogenous ROS. These results suggested that different ROS level in different cell cycle phases of NB4 cells might determin the selective induction of G2/M apoptosis and the cells‘ susceptibility to apoptosis by As2O3.